We propose to tackle the challenge (06-HL-103) of providing enabling technologies directed towards developing novel quantitative imaging approaches for the measurement of coagulation enzyme function in the intact mouse to improve basic understanding of cellular interactions, the biological pathway for coagulation and its regulation in flowing blood. The intravital fluorescence microscopy approach for examining thrombus formation following laser-induced damage in the cremaster arteriole of the mouse has opened new avenues for quantitative studies of thrombus formation in the blood of a living organism. However, the approach has relied on indirect measures employing fluorescent antibodies to select reactants. Its full promise has been limited by the availability of reagents and approaches for the direct measurement of the individual steps of coagulation. We propose to exploit our established expertise in the biochemistry of the coagulation reactions coupled with an array of well-characterized site-specific fluorescent derivatives of the clotting proteins with defined properties to develop enabling technologies directed towards sensitive and quantitative measurements of coagulation enzyme complex assembly and function at the site of the growing thrombus. We propose approaches designed to examine the spatial relationship between coagulation enzyme localization and the evolving thrombus. We also propose to utilize this new technology to examine the contributions of cells present at the site of injury to differentially support coagulation enzyme function. The enabling aspect of our studies will be enhanced by the expansion of the repertoire of site-specific fluorescent coagulation protein derivatives to include a series of recombinant mouse proteins. Recognizing the widespread interest in the use of genetically manipulated mouse models in coagulation, we submit that our strategies will equip the field with the enabling technologies to move the intravital imaging approach to the next level and permit widely applicable studies to decipher the regulation of the blood coagulation pathways in vivo. CHOP contributes substantially to the local economy. In 2008, CHOP's operations created and supported over 16,882 jobs in the region, and CHOP's total economic impact was over $2.01 billion. Moreover, through a combination of private donations, NIH funding, and allocations from its hospital operations, CHOP receives more total research support than any other children's hospital in the United States -- $180 million in fiscal year 2007-2008. The current proposal will create 2 jobs and provide partial support for the retention of jobs for 2 investigators. The formation of a blood clot at the site of injury results from a series of clotting reactions that occur in concert with blood cells and cells lining the blood vessel in flowing blood. We seek to solve the challenge of imaging the blood clotting reactions in the living organism by the development of novel fluorescent approaches and reagents based on knowledge of the biochemistry of coagulation. This enabling technology will provide the wherewithal for new insights into how blood clots form under normal conditions and in life-threatening human diseases. PUBLIC HEALTH RELEVANCE: The formation of a blood clot at the site of injury results from a series of clotting reactions that occur in concert with blood cells and cells lining the blood vessel in flowing blood. We seek to solve the challenge of imaging the blood clotting reactions in the living organism by the development of novel fluorescent approaches and reagents based on knowledge of the biochemistry of coagulation. This enabling technology will provide the wherewithal for new insights into how blood clots form under normal conditions and in life-threatening human diseases.